The following prior art references are considered to be relevant for an understanding of the invention:    1. Burt, A. & Koufopanou, V. Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr Opin Genet Dev 14, 609-615 (2004).    2. Stoddard, B. L. Homing endonuclease structure and function. Q Rev Biophys 38, 49-95 (2005).    3. Paques, F. & Duchateau, P. Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7, 49-66 (2007).    4. Arnould, S. et al. Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 371, 49-65 (2007).    5. Smith, J. et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res 34, e149 (2006).    6. Scalley-Kim, M., McConnell-Smith, A. & Stoddard, B. L. Coevolution of a homing endonuclease and its host target sequence. J Mol Biol 372, 1305-1319 (2007).    7. Kurokawa, S. et al. Adaptation of intronic homing endonuclease for successful horizontal transmission. Febs J 272, 2487-2496 (2005).    8. U.S. Pat. Nos. 6,528,313 and 6,528,314, European patent EP 419 621 and Japanese patents JP 3059481, JP 3298842 and JP 3298864.
Gene therapy aims to cure diseases by treating their genetic basis rather than their manifestations. It entails the delivery of corrective genes into affected cells in order to replace, inhibit, correct or compensate for the expression of a disease causing allele. The great promise of gene therapy is to provide a remedy for illnesses that are otherwise difficult to address, such as congenital genetic disorders, neurodegenerative diseases, viral infections and cancer. However, after years of research, two main challenges still stand in the way of wide and successful gene therapy applications. First, the vector carrying the corrective gene must be delivered to the appropriate tissues or cell types and only to them, in order to avoid toxic side effects. Second, when the corrective gene has entered the cell, it must be expressed in a controlled manner, namely, at the correct time, to the appropriate extent and without disturbing the due expression of other important genes. Controlled expression can best be achieved by replacing or correcting the mutated gene at its native location, under the indigenous promoter, where both cis and trans regulators can exert their normal effects. This form of precise correction or replacement is called gene-targeting. In addition to the above medical utilities, gene targeting can also be used for biotechnological enterprises such as crop improvement and for research undertakings such as the engineering of knockout mice strains that allow scientists to model human diseases and test potential remedies.
Transfection of human cells by vectors carrying a corrective gene very rarely results in gene targeting. These rare events are attributed to spontaneous homologous recombination (HR) between the vector-borne gene and the endogenous allele. There are several ways to increase the rate of HR; by far the most effective of which is the induction of a site specific double strand break (DSB). Such DSBs have been shown to raise the frequency of gene targeting by as much as three orders of magnitude. However, induction of a unique DSB is challenging due to the shear size of the human genome (about 3*109 base pairs (bp)). For example, a restriction enzyme with an 8 by long target sequence will cleave the human genome approximately 3*109/48≈45,776 times. Such excessive or non-specific cleavage may result in cell death or worse, in genomic instability leading to malignant transformation. There are two major approaches to the challenge of introducing unique DSBs into the human genome. The first approach entails the design of chimeric proteins consisting of a non-specific endonuclease domain linked to a combination of DNA binding domains; the latter are typically zinc finger domains and the chimeras are zinc finger nucleases or ZFNs. ZFNs have been shown capable of inducing gene targeting in human cells. However, much concern has been raised regarding their possible toxicity.
The alternative approach advocates the use and manipulation of naturally occurring site-specific DNAases having long target sequences, namely homing endonuclease genes or HEGs. HEGs are a large and diverse class of site-specific DNAases found in Archaea, Eubacteria and lower eukaryotes, and in their respective viruses. The lengths of HEG target sequences range between 14-40 bp. Furthermore, these targets are not stringently defined. Cleavage is tolerant to some base-pair substitutions along the target sequence. This has raised hopes that at least some HEGs can introduce unique DSBs in desired loci of the human genome. However, only a few hundred HEGs have been annotated to date, and only a few dozen of which have been experimentally characterized. Chances are therefore slim for finding within this limited collection a HEG suitable for gene targeting of a desired gene. One possible way to circumvent this limitation is by attempting to shift the target specificity of a given HEG to make it capable of cleaving a desired sequence (e.g. one that is found within a disease related gene). This has been done with considerable success using a combination of directed enzyme evolution and rational design. Engineered HEGs have been manufactured capable of cleaving XPC (deficient in Xeroderma Pigmentosum), IL2RG (deficient in X-linked SCID—severe combined immunodeficiency), Rag1 (deficient in autosomal recessive SCID) and the tumor suppressor gene p53. Despite its achievements, HEG-engineering is an inherently limited approach; using directed evolution and rational design one can only alter target specificity up to a certain extent. Therefore, for HEG mediated gene targeting to become a common medical practice, the arsenal of target sites must be dramatically extended by the discovery of many more naturally occurring HEGs.
A homing endonuclease (HE) cleaves a long (14-40 bp), rather specific, DNA target sequence. FIG. 1 shows schematically the expression of a HEG, and the activity of a HE. A HEG 2 is found within a self-splicing intron 4 or intein (not shown) within a gene 6. The active HE 8 is produced following splicing of the mRNA transcribed from the gene 6, or splicing of the protein translated from the mRNA. The HE 8 recognizes and cleaves a target sequence 10 in a “vacant allele” 12 of the gene 6 which lacks the intron 4 or the intein. The HE 8 can then promote the insertion of a copy of the intron 4 or the intein into the vacant allele 12 by homologous recombination (homing) or reverse transcription (retro-homing)1,2. Thus, the HE target site 10 also marks the insertion site of the intron 4.
HEs have been utilized in gene targeting procedures where the introduction of site-specific double-strand-breaks facilitates gene correction, disruption or insertion at a locus of choice3. U.S. Pat. Nos. 6,528,313 and 6,528,314, European patent EP 419 621 and Japanese patents JP 3059481, JP 3298842 and JP 3298864 disclose use of homing endonucleases in gene targeting.
For a HE capable of cleaving only its cognate target, straightforward probabilistic considerations would render HE-mediated gene targeting a virtual impossibility. For example, a 25 by long target sequence would be expected to be found at random every 425≈1015 bp. It has a one in a million chance of being found anywhere in the human genome, let alone in a medically important gene.
Only a few hundred HEs have been identified, and only a few dozen of them have been characterized experimentally. The chances are small of finding within this limited collection a HE capable of cleaving a selected nucleotide sequence (e.g. a sequence found within a disease related allele of a gene). One possible way to overcome this limitation is by attempting to change the target specificity of a given HE so that the GH can cleave the selected sequence. This has been done using a combination of directed enzyme evolution and rational design. HEs have been engineered capable of cleaving XPC (deficient in Xeroderma Pigmentosum), IL2RG (deficient in X-linked SCID-severe combined immunodeficiency), Rag1 (deficient in autosomal recessive SCID) and the tumor suppressor gene p53. However, HE-engineering is an inherently limited approach in that directed evolution and rational design one can only alter the target specificity to a limited extent.
Burt, and Koufopanou1 and Stoddard2 have reported HEs capable of cleaving base-pair sequences differing from their cognate target site by at most a few base pair substitutions1,2. Base-pair substitutions at non-conserved positions are, in general, better tolerated by the HE than base pair substitutions at highly conserved positions6,7. In particular, HEs are more tolerant of synonymous substitutions than they are of non-synonymous substitutions6,7.